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Derek Lowe's commentary on drug discovery and the pharma industry. An editorially independent blog from the publishers of Science Translational Medicine. All content is Derek’s own, and he does not in any way speak for his employer.

Making Cells Do Your Work (Also, The Revenge of the Yeast)

Enzymes do things we can’t do. That’s one of the facts of life in organic synthesis, and it’s likely to be true for quite a while. (We can do things that enzymes can’t do, to be sure, but in many cases that’s probably because enzymes have never yet seen a need to do them). Cells, most especially, can do chemical transformations that we can’t do, because there the various enzymes are in their happy native environments, and entire enzymes systems are assembled and ready to get to work.

Biotechnology takes great advantage of this through recombinant cells, and any number of proteins (and protein-based drugs) are produced via live cell culture. Small molecules, not so much. The protein-making machinery is mighty and ubiquitous, but the small-molecule-making pathways, once you get away from the common molecules of life, tend to be little bespoke cottage industries inside a cell. Some natural-product-based compounds and starting materials are made this way (antibiotics, the starting material for taxol, etc.), but even these are taking advantage, for the most part, of specific pathways that the cells already had up and running, rather than having them make anything different.

You can always go in and try to modify and engineer enzymes, but this is already enough of a project with isolated enzymes, much less in living cells. An easier way to extend the concept is to give the cells other substrates and see what they make of them. Some interesting “unnatural products” have been made this way, but a more common use is to take advantage of a particular enzyme pathway, in most cases a chiral reduction. Live yeast has been a favorite system for such reactions for many decades now, and it’s one of those transformations in the “When it works, it works” category. When it doesn’t work, you can always try a different strain of yeast, and plenty have been developed over the years. You can buy collections of them, and collections of some of the isolated enzymes to see what might work for you.

The ideal would be to do such screening in as close to a single-cell manner as possible. That would give you some variety in screening, but it’s a significant technical challenge – isolating the cells, keeping them happy, getting the substrate to them, analyzing the products on such a small scale. Here’s a paper that’s working towards that goal, though: they’re work down to the several-hundred-cell level, using E. coli cells engineered with an Aspergillus enzyme. It’s done in an integrated microfluidic chip, with an ingenious built-in electrophoresis step, and a deep-UV fluorescence detector. None of these would be your first choice if you were doing the reaction in a flask, of course, but this is just the sort of thing that’s going to have to be worked through on the way to the single-cell world, and I’m glad to see people making the effort. You could imagine, eventually, growing up a batch of some test organism with an induced high mutation rate, screening huge numbers of individual cells, and quickly arriving at a good enzymatic starting point. Speed the day!

I can’t talk about yeast reductions without telling a story, though. Back when I was in graduate school, we had a Japanese post-doc in the lab who needed to do one of these on pretty large scale to get a chiral starting material. He had, though, apparently never done a yeast reaction, and had probably never seen yeast in action at all. At least, that’s what we gathered from what happened. I was down in that lab using the analytical balance (the only one for the whole group – those were the days), when I heard our normally quiet Japanese guy suddenly start making plenty of noise. Unless I missed my guess completely, those sounded like vigorous Japanese curses and sounds of alarm.

What he’d done, it turned out, was to go get a four-liter Erlenmeyer, dump in a goodly heap of dry baker’s yeast, pour some lukewarm water into it, and then give it a good slug of sugar to really get it going. It got going. When I came around to his hood, the post-doc was staring in dismay at a yeast volcano: an apparently endless stream of tan foam was swiftly overflowing the Erlenmeyer and spreading out into his hood. Still shouting in displeased Japanese, he located a bucket and put the flask into it, but that was only a holding measure. The foam kept on coming, and was obviously going to fill the bucket before finally winding down. By this time, he’d attracted a crowd of appreciative onlookers – the last thing he wanted, I’m sure – and we were encouraging him with helpful suggestions like adding more sugar, putting some green food coloring in the flask and calling the campus newspaper, and so on. He finally groaned, poured the lot down the sink, and went home.

A few days later, though, he spoke at group meeting, and had apparently gotten over the trauma. “I try yeast reduction”, he told he group, grinning. “But yeast is very dangerous.” He reached into a bag he had with him, and pulled out a dinner roll: “However, it makes very good bread!”

14 comments on “Making Cells Do Your Work (Also, The Revenge of the Yeast)”

Had he been a homebrewer, he would have known what to expect. Brewing my own beer has made me a better chemist. Well, not really, but it is the most fun you can have doing chemistry/biochemistry at home. Unlike work chemistry, both mistakes (most non-horrendous ones anyway) and successes can be relatively enjoyable.

I had similar volcano experience with learning how to do a yeast reduction step. Of course for a week or so visitors did not complain about the typical smelly organic lab and labeled us the Bakery. Like too much of anything after a few hours in the lab most people had to go out for fresher air. Part of the problem is when OrgSyn people try to do biochem will simply attempt to apply the tools we are most familiar with and not think things through clearly. Erlenmeyer is an extremely poor choice as the narrowed neck acts as a cannon tube whereas the biochemists I saw either used large beakers, buckets or oversized jugs to contain the foaming potential.

Back in the late 80’s, I took a short course given by Tomas Hudlicky for a week at the University of Florida, that was well worth it in my opinion. It really opened ones eyes as to what a synthetic chemist could do in his own lab with cells, and even some commercially available isolated enzymes. We did much more than sit in lectures. We also had a half day each day of lab work, were we learned techniques to doing these Biotransformations in using ordinary laboratory glassware. Also – I have used antifoaming agents successfully to control these sorts of things as well. This just adds more tools to the tool box. Once upon a time – the larger pharma companies had some substantial experience in doing these things on very large scales, and usually had fermentation groups feeding interesting molecules to yeast and bacteria.

I’ve proofed yeast over 1000 times, and my rule is the container must be at least 5X larger than the contents. Even then, I’ve had a couple overflows, and this was always with minimal sugar. If you don’t know how much sugar that is, I’d recommend 10X larger. If possible use something wide like a beaker or a jar, not something with a neck in it like an Erlenmeyer flask unless you’re sure you’ve got enough headspace. Rate of growth varies strongly with ambient temperature, so what works fine on a cool day may yield surprises on a warm day. To goose my yeast culture without killing it, I place my jar on top of the hot spot on my Mac iBook. On a cold day, that provides just the right amount of heat input.

My wife is a veterinarian. One of the emergency calls she talks about is a dog that ate a whole bowl of yeast dough set out to rise. Dog was in serious shape as it continued to rise in his stomach with nowhere to go.

I would have loaded 100 mg 3-bromopyruvate into a 10 mg syringe and injected it into the yeast volcano. The only thing cooler than an out of control yeast volcano is showing a floor of PhDs how to instantly shut down cellular respiration.

If that didn’t work out you’d have 100 mg of aerosolized 3-BP to contain.

Brings back memories. The first step of my graduate total synthesis was a yeast reduction of a 2-dialkyl-1,3-pentadione. Used to run multiples reactions on a 10 g (substrate) scale simultaneously. My lab smelled like a brewery, which was quite pleasant. Unfortunately, during the workup, which involved lysis of cells with ethyl acetate, was not so olfactorally pleasant – especially with a hood that were originally installed in 1928 – followed by filtration of the sticky, slimey cellular debris. All unpleasantness aside, it was an efficient means to produce my chiral starting material.